1. Field of the Invention
This invention relates to a DNA fragment, a recombinant DNA suitable to produce a breed of plant such as a maize or rice plant having a resistance to low temperatures, and a transformed plant.
2. Description of Related Art
From studies so far, it has been found that there is a significant relationship between the resistance of a plant to a low temperature and the degree of unsaturation of the fatty acids constructing a biomembrane thereof.
The inventors experimentally indicated that a transformed tobacco plant comes to have a higher resistance against low temperatures when a fatty acid unsaturating enzyme gene FAD7 derived from an arabidopsis is highly expressed in the plant.
When the production of a certain protein in a plant cell is required, a promoter operating constitutively and having a high promoter activity has been used. Such a promoter operates continuously, which is unnecessary in the plant.
For instance, a constitutive promoter is also employed when a protein, which gives a plant resistance to low temperatures, is expressed. Therefore, even at ordinary temperatures, the protein, which gives the plant resistance to low temperatures, is forced to be unnecessarily expressed, causing unfavorable results.
For such reasons, the development of a site-specific and inducible promoter has been required. Particularly, it is required that the expression of a specific gene is strengthened only at low temperatures, in order to produce a breeding intermediate mother body in which a gene expressing an enzyme for unsaturating a fatty acid and other protein genes contributing to a low temperature resistance are inducibly expressed in response to low temperatures, or in order to make possible the production of an unstable functional protein using a plant cell.
It is, therefore, an object of the invention to produce a promoter capable of strengthening the expression of a fatty-acid-unsaturating-enzyme gene and other protein genes contributing to a low temperature resistance in response to low temperatures.
Another object of the invention is to produce a breed of plant having a low temperature resistance using the above promoter.
Still another object of the invention is to utilize a promoter in an inducible system of a functional protein in a plant cell, said promoter being specific to low temperatures.
According to a first aspect of the invention, there is the provision of a DNA fragment comprising a base sequence (a) or (b) described below:
(a) The base sequence referred to as nucleotide numbers 1-3794 of SEQ ID NO;1;
(b) The base sequence (a) a part of which is deleted or substituted by another base sequence, or to which another base sequence is added.
According to an aspect of the invention, there is the provision of a recombinant DNA and a transformed plant having a low temperature resistance and said plant is transformed with said recombinant DNA.
According to a second aspect of the invention, there is the provision of a DNA fragment with a promoter activity and with a responsive property to low temperatures, which comprises a base sequence (c), (d), or (e) described below:
(c) The base sequence referred to as nucleotide numbers 1-2797 of SEQ ID NO:1;
(d) A part of the base sequence (c) with a promoter activity and with a responsive property to low temperatures;
(e) The base sequence (c) or (d), a part of which is deleted or substituted by another base sequence, or to which another base sequence is added.
According to another aspect of the invention, there is the provision of a recombinant DNA which includes the DNA fragment comprising the base sequence (c), (d), or (e) described above, and a transformed plant transformed with the recombinant DNA and which phenotypically expresses a specific protein in response to low temperatures.
According to a third aspect of the invention, there is the provision of a DNA fragment with a promoter activity, which comprises a base sequence (f), (g), or (h) described below:
(f) The base sequence referred to as nucleotide numbers 1-2271 of SEQ ID NO;1;
(g) A part of the base sequence (f) with a promoter activity;
(h) The base sequence (f) or (g) a part of which is deleted or substituted by another base sequence, or to which another base sequence is added.
According to a forth aspect of the invention, there is the provision of a DNA fragment with a responsive property to low temperatures, which comprises a base sequence (i), (j), or (k) described below:
(i) The base sequence referred to as nucleotide numbers 2272-2797 of SEQ ID NO:1;
(j) A part of the base sequence (i) with a responsive property to low temperatures;
(k) The base sequence (i) or (j), a part of which is deleted or substituted by another base sequence, or to which another base sequence is added.
Since the rice low temperature inducible gene lip19 or the maize low temperature inducible gene mlip15 codes a DNA binding factor, they are considered to be a gene controlling transcription of other gene groups induced or repressed under low temperature stress. The inventors have clarified a functional unit of a low temperature inducible promoter in the maize""s mlip15 gene and have clarified that the functional unit also operates in a transformed plant.
The inventors have isolated a genomic clone of the maize""s mlip15 gene by a common method. After determination of the nucleotide sequence, it became clear that the gene does not include an intron (see: Sequence number 1 in Sequence list)
In the gene mlip15, 2.8 kb of the nucleotide sequence (0.6 kb of a nontranslated region at 5xe2x80x2-end and 2.2 kb of a nucleotide sequence linking at upstream thereof, in a mlip15 cDNA) and 2.2 kb of the nucleotide sequence (the nucleotide sequence remained after subtracting 0.6 kb of the nontranslated region at 5xe2x80x2-end from the 2.8 kb of the nucleotide sequence) are respectively fused with a xcex2-glucuronidase gene as a reporter gene to make recombinant DNAs.
Each recombinant DNA is introduced into a callus derived from a rice""s scutellum by using a particle gun. The result is that the former (the 2.8 kb nucleotide sequence) maintains a reactivity for low temperatures but the latter (the 2.2 kb nucleotide sequence) loses it. Therefore, it has been clarified that the 2.8 kb fragment containing 0.6 kb of the nontranslated region at 5xe2x80x2-end has a promoter function responsive to low temperatures.
In the invention, as a vector used for producing a recombinant DNA, a plasmid can, for example, be employed. Further, as a plant in which to introduce the recombinant DNA, a useful cultivating monocotyledon such as maize, rice, wheat, barley, oat, Italian millet, or Japanese millet is preferably used.
A protein produced by induction of the promoter according to the invention includes an xcfx89-3-fatty acid unsaturating enzyme etc.
Moreover, the invention includes a base sequence where one or several nucleotides of which are deleted or substituted by another base sequence, or to which another base sequence is added, if it maintains the promoter function according to the invention.